Irritable bowel syndrome (IBS) is estimated to affect up to 15% of the population in the USA, with negative effects on quality of life and annual direct and indirect health care costs of ~30 billion U.S. dollars. Pain is the characteristic symptom of IBS and the main reason for patient visits to gastroenterologists. Accumulating evidence suggests that heightened peripheral sensory input from colorectal afferents (i.e., afferent sensitization) is necessary for the persistence of IBS-related pain and hypersensitivity. The goal of the proposed research is to investigate molecular/neurochemical identities ('chemotype') and histological locations of functionally distinct afferent classes in the colorectum, from which information about mechanisms of afferent sensitization that contribute to colorectal hypersensitivity will be revealed. Specifically, a novel ex vivo preparation of the mouse colorectum with nerves and spinal cord in continuity will be used to couple functional study of afferent electrophysiology with chemotype evaluation and afferent ending anatomy. Studies will be done in the context of long-term models of visceral hypersensitivity induced by intracolonic treatment with zymosan or trinitrobenzene sulfonic acid (TNBS), respectively. Three specific aims are proposed. Specific Aim 1 will characterize chemotype of functionally distinct classes of afferents in the pelvic nerve (PN) colorectal innervation. Specific Aim 2 will determine the histological location and morphological features of functionally distinct classes of afferents in te PN colorectal innervation. Specific Aim 3 will explore the underlying mechanisms for enhanced neural encoding of colorectal afferents using computational simulation. Specific Aim 4 will explore the possible translation of outcomes from the foregoing studies into clinic targets for management of colorectal pain and hypersensitivity by studying human intestinal afferents in vitro. The combined functional, chemotypical, and morphological approaches will allow generation of new information about channels/receptors that contribute to afferent sensitization and, for the first time, reveal histological/morphological features of colorectal receptive endings Experimental data will be used to construct and validate a computational simulation, which will allow exploration of the putative mechanism(s) for sensitization in each functionally distinct afferent class. The outcomes of the proposed experiments have the potential to guide strategies for development of pharmacological compounds that possess colorectal afferent sub-class specificity.